Abstract

To investigate the role of estrogen in flow-induced dilation (FiD) in nitric oxide (NO) deficiency, FiD was examined in isolated gracilis arterioles of ovariectomized (OVX) and OVX rats with estrogen replacement (OVE). Both groups of rats were treated chronically withNω-nitro-l-arginine methyl ester. Plasma concentration of NO2/NO3 was reduced in both groups. Plasma concentration of estradiol was lower in OVX than in OVE rats. FiD was similar in vessels of the two groups; calculated wall shear stress and basal tone were significantly greater in OVX vs. OVE rats. Indomethacin did not affect FiD in vessels from OVE rats but abolished dilation in vessels from OVX rats. Valeryl salicylate or NS-398 inhibited FiD by ∼50%, whereas their simultaneous administration eliminated the response in arterioles from OVX rats. In vessels from OVE rats, miconazole or charybdotoxin eliminated FiD. Thus, in NO deficiency, prostaglandins derived from both cyclooxygenase isoforms mediate FiD in gracilis arterioles of OVX rats. Estrogen replacement switches the mediation, showing dependence on endothelium-derived hyperpolarizing factor in the arterioles of OVE rats.

ovariectomy

estrogen replacement

potassium channels

arterioles

endothelium-derived hyperpolarizing factor

nitric oxide

endothelial cells contribute to the control of vascular tone through the synthesis and/or release of vasoactive agents that affect the contractile activity of the underlying smooth muscle. This regulatory function of the endothelium can be modulated by pharmacological agents and mechanical forces, such as pressure and shear stress. As a physiologically relevant stimulus in vivo, shear stress mediates vascular dilator responses by triggering the release of endothelial nitric oxide (NO), prostaglandins, and endothelium-derived hyperpolarizing factor (EDHF) (19, 20, 23). The contributions of these factors to the mediation of flow- and/or shear stress-induced dilation are dependent not only on the different species and vascular beds studied but also on their interactions (3, 4, 23). Our laboratory's recently published studies revealed a gender difference in the endothelial mediators eliciting flow-induced dilation in NO-deficient states (13, 28, 31). These studies showed that flow-dependent responses of arterioles are mediated exclusively by EDHF in female endothelial NO synthase (eNOS)-knockout mice and by prostaglandins in male littermates. This gender-dependent compensation for NO deficiency was also demonstrated in rats that were chronically treated withNω-nitro-l-arginine methyl ester (l-NAME). These congruent findings obtained from two different species and models provide evidence to support our conclusion that arteriolar responses evoked in the absence of NO are indeed gender dependent in nature, although the specific mechanisms by which endothelial cells sense a change in shear stress and convert it into biochemical signals to account for the release of specific mediators are still unknown. In line with the foregoing, we hypothesized that estrogen is responsible for the gender-specific regulation of flow-induced dilation of arterioles lacking eNOS. Thus we conducted experiments on skeletal muscle arterioles of l-NAME-treated female rats that had been ovariectomized (designated OVX) or ovariectomized and given estrogen replacement (designated OVE).

METHODS

Animals.

Seven-week-old female Wistar rats (Charles River Laboratories, Wilmington, MA) were ovariectomized (14-17) and then received l-NAME in drinking water (50 mg/100 ml) for 4 wk. During the period of l-NAME treatment, rats received injections of either 17β-estradiol benzoate subcutaneously (50 μg/kg in sesame oil every 48 h) (OVE rats) or the vehicle (OVX rats). Systolic blood pressure was monitored with a tail-cuff method. All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the guidelines of the National Institutes of Health and the American Physiological Society for the use and care of laboratory animals.

Measurement of plasma estradiol and nitrite/nitrate.

Ten milliliters of blood were withdrawn from rat abdominal aorta with a 10-ml syringe containing 0.1 ml of heparin (1,000 UPS U/ml) before the rats were killed. The blood sample was centrifuged immediately (3,000 rpm at 4°C for 30 min) to obtain the plasma, which was then divided into two parts for the measurement of estradiol and nitrite/nitrate (NO2/NO3), respectively.

Plasma estradiol concentration was measured by a radioimmunoassay with a double-antibody estradiol kit (Diagnostic Products) (15,17). Plasma NO2/NO3 was measured by using a fluorometric assay (31).

Experimental procedures.

Experiments were conducted on isolated gracilis muscle arterioles of rats. The dissection of muscle, isolation of vessels, and experimental setup have been described previously (14-18, 31). Changes in diameter of arterioles in response to increases in flow were studied at 80 mmHg of intraluminal pressure. Perfusate flow was increased from 0 to 25 μl/min, in 5 μl/min steps.

In the first series of experiments, a role of prostaglandins in the mediation of flow-induced dilation was assessed by using indomethacin (Indo, 10−5M), a nonselective inhibitor of cyclooxygenase (COX).

In the second series of experiments, the specific roles of COX isoforms (COX-1 and COX-2) in the prostaglandin-mediated flow-induced dilation were evaluated by performing the experiments before and after administration of valeryl salicylate (VSA, 3 × 10−3M) andN-[2-(cyclohexyloxy)−4-nitrophenyl]-methanesulfonamide (NS-398, 10−5 M), specific inhibitors of COX-1 and COX-2, respectively. The inhibitors were given in different sequences, alone and in combination.

In the third series of experiments, the role of metabolites of cytochrome P-450 (CYP) in flow-induced dilations was assessed by using miconazole (MCZ, 2 × 10−6 M), an inhibitor of CYP/epoxygenase.

Passive diameter.

At the conclusion of each experiment, the suffusion solution was changed to a Ca2+-free solution containing 1 mM EGTA. Vessels were incubated for 10 min to reach maximal diameter at 80-mmHg perfusion pressure.

Chemicals.

All chemicals were obtained from Sigma Chemical (St. Louis, MO). ChTX was dissolved in saline. Indo, MCZ, VSA, and NS-398 were dissolved in DMSO at a concentration of 10−1 M (for Indo), 10−2 M (for MCZ and NS-398), and 3 × 10−3 M (for VSA) and further diluted with physiological salt solution. The highest concentration of DMSO in the chamber was 0.1% (vol/vol), which had no significant effect on the vessel tone. The 17β-estradiol benzoate was dissolved in pure ethanol (5 mg/ml) with sesame oil as vehicle.

Calculations and statistics.

Passive diameter was used to assess the active tone (% of passive diameter) generated by arterioles in response to intravascular pressure and to normalize the changes in diameter in response to increases in flow in each vessel. Wall shear stress was calculated by the equation 4ηQ˙/πr3, where η is the viscosity of the perfusion solution (0.007 poise at 37°C), Q˙ is the perfusate flow, and r is the vessel radius. Data are presented as means ± SE; n is the number of rats. Statistical significance was calculated by repeated-measures two-way ANOVA followed by Tukey-Kramer's multiple-comparison test. Student'st-test was also used, as appropriate. Significance level was taken at P < 0.05.

RESULTS

Table 1 summarizes the changes in body weight, uterine weight, the uterine-to-body weight ratio, blood pressure, and plasma concentrations of estradiol and NO2/NO3 in two groups of rats. The uterus weights of OVX rats were significantly lower than those of OVE rats. In contrast, the body weights of OVX rats were significantly greater than body weights of OVE rats. As a result, the ratio of uterus weight to body weight was significantly less in OVX compared with that of OVE rats. Plasma concentration of estradiol, which was negligible as a result of ovariectomy, was normalized by estrogen replacement. Also, as a function of l-NAME treatment, plasma concentration of NO2/NO3 was reduced and was accompanied by a significant elevation of blood pressure.

Characteristics of arterioles of gracilis muscle from the two groups of rats are summarized in Table 2. The active diameter was significantly smaller in arterioles of OVX rats vs. those of OVE rats, but their passive diameters were similar. Active diameters, as a percentage of the corresponding passive diameters, indicated a significantly greater basal tone in arterioles of OVX rats compared with those of OVE rats.

Figure 1 shows normalized diameter (A) and wall shear stress (B), as a function of perfusate flow, in arterioles of both groups of rats. The changes in diameter of arterioles, in response to step increases in perfusate flow, were not significantly different in the two groups of rats. However, increases in flow elicited significantly greater increases in shear stress, at each flow rate, in arterioles of OVX vs. OVE rats.

The endothelial mediators responsible for flow-induced dilations of arterioles of OVX rats are illustrated in Fig.2, showing that Indo, which did not affect the basal tone of arterioles from either group of rats, abolished the dilator responses to flow (Fig. 2A). In a separate group of experiments, the specific roles of COX-1 and COX-2 in the mediation of Indo-sensitive flow-induced dilation were tested by using VSA and NS-398, respectively. Each inhibitor alone significantly inhibited flow-induced dilation by ∼50%, and a combination of both inhibitors essentially eliminated the responses (Fig. 2, Band C).

Normalized diameter of gracilis muscle arterioles ofl-NAME-treated OVX rats (n = 5–8 for each group), as a function of perfusate flow, in the control condition and after administration of indomethacin (Indo, 10−5 M;A) or valeryl salicylate (VSA, 3 × 10−3M) or NS-398 (10−5 M) in different sequences, alone and in combination (B and C). Values are means ± SE. *Significant difference between the 2 curves calculated by repeated measures of two-way ANOVA, P < 0.05.

The endothelial mediators responsible for flow-induced dilations of arterioles of OVE rats are shown in Figs. 3 and 4. Indo, although eliminating flow-induced dilations in arterioles of OVX rats, had no effect on the responses of OVE rat arterioles (Fig.3A). However, the Indo-resistant, flow-induced dilations were abolished by MCZ (Fig. 3), indicating a CYP-dependent response. Furthermore, when the arterioles were treated with ChTX, a blocker of Ca2+-dependent K+ channels, a target of CYP metabolites/EDHF in smooth muscle (13, 31), flow-induced dilation was abolished (Fig. 4A). However, ChTX did not affect the responses in arterioles of OVX rats (Fig.4B).

DISCUSSION

The present study confirms our hypothesis that the gender difference in the adaptation to the lack of NO is estrogen dependent. In l-NAME-treated OVX rats, flow-induced dilation is solely mediated by prostaglandins, a response that mimics that observed in NO-deficient arterioles of male rats and mice (28, 31). An upregulation of COX-2, together with prostaglandins derived from COX-1, seems to be responsible for the mediation of the response. Estrogen replacement causes a switch of the prostaglandin mediation to an EDHF-mediated response, recovering entirely the profile of the response observed in NO-deficient arterioles of female rats and mice (13,31).

To single out the specific role of estrogen in the gender differences observed in the mediation of flow-induced dilation when NO is absent,l-NAME-treated female rats were ovariectomized and then received hormone replacement treatment with 17β-estradiol or injections of the vehicle. The significant reduction in uterine weight and plasma concentration of estrogen and the reversal of this reduction in animals receiving estrogen demonstrate the effectiveness of ovariectomy and estrogen replacement therapy. The significant increases in blood pressure and reduced plasma NO2/NO3level are indicative of NO deficiency caused by chronic treatment withl-NAME (Table 1).

Basal tone of arterioles of NO-deficient OVX and OVE rats.

Ovariectomy significantly enhanced the basal tone of arterioles of OVX rats, due to a decrease in their active diameter but without affecting their passive diameter. Estrogen replacement decreased the basal tone, suggesting that the chronic presence of circulating estrogen is necessary for the maintenance of a reduced basal tone of arterioles (Table 2). We previously demonstrated that, due to the presence of estrogen, arterioles from females have a lower basal tone than those from males, which is related to an enhanced release of endothelium-derived NO (14, 16). In the present study, the reduced basal tone of arterioles from estrogen-treated rats persists even in the absence of NO, indicating that non-NO-dependent mechanisms are also involved. Because neither Indo nor MCZ affected the basal diameter of arterioles in the present study, we suspect that the estrogen-related attenuation of arteriolar tone in NO deficiency may not be dependent on the presence of endothelium.

Flow- and shear stress-induced dilation in arterioles of NO-deficient OVX and OVE rats.

We previously demonstrated a greatly enhanced NO-contribution to flow-induced dilation in arterioles of normal female and OVE rats, compared with that in arterioles of male and OVX rats (15, 17,18). In the present study, conducted on vessels ofl-NAME-treated OVX and OVE rats, however, increases in perfusate flow dilated the arterioles to an equal degree (Fig.1A). On the other hand, the physiological relevance of estrogen replacement is indicated by a significantly reduced wall shear stress, at each flow step, (Fig. 1B) in arterioles of OVE rats, suggesting that in vessels of these rats a lower shear stress is required than in those from OVX rats to achieve a dilation of similar magnitude.

Mediation of dilation to flow in arterioles of NO-deficient OVX rats.

In adapting to the lack of NO and estrogen, flow-induced dilation of arterioles from OVX rats is exclusively prostaglandin dependent, as indicated by the elimination of the responses with Indo (Fig.2A). These results are identical to those observed in male mice and rats, in which eNOS is absent due to the genetic ablation of the eNOS gene (28) or chronic treatment withl-NAME (31). The upregulation of prostaglandin synthesis in response to the absence of NO activity has been well documented (11, 28). More specifically, however, we demonstrate here that the compensatory upregulation of prostaglandin synthesis involves inducible COX (COX-2), since NS-398 inhibits the portion of response that is otherwise mediated by NO in normal male rats and mice (28, 31). On the other hand, NS-398 has no effect on the Indo-sensitive portion of flow-induced dilation in normal males, but VSA does. In keeping with the present findings, expression of the COX-2 gene in response to shear stress has also been demonstrated (26) in human umbilical vein endothelial cells. In addition, it was reported that vanadate, an inhibitor of protein-tyrosine phosphatase, elicited the expression of COX-2 mRNA and consequently increased corresponding protein levels in human umbilical vein endothelial cells. This vanadate-induced enhancement of expression of COX-2 mRNA was abolished by tyrphostin-47, an inhibitor of protein-tyrosine kinases (12). In this context, we previously found that flow-induced dilation in skeletal muscle arterioles is tyrosine protein phosphorylation dependent (29). The question then arises as to why this compensatory activity, in response to NO deficiency, depends specifically on COX-2. The literature regarding the “cross-talk” between the NOS and COX pathways is divided with respect to whether NO activates or inhibits prostaglandin production (1, 10, 27). Recent evidence shows that NO exerts divergent effects on the constitutive and inducible COX isoforms, potentiating COX-1 but inhibiting COX-2 (6). This study reports that exposure of resting cells to NO enhances the production of PGE2, which was inhibited by Indo but not by NS-398. In contrast, exposure of lipopolysaccharide-stimulated cells to NO inhibited PGE2production, which associated with a decrease in COX-2 expression and nitration of the enzyme, which interferes with its catalytic activity (9). Thus the divergent effects of NO on the COX isoforms may explain why COX-2 becomes functional when NO synthesis is absent. Similar findings in mesenteric arteries ofl-NAME-treated rats indicated an overproduction of prostaglandins in response to shear stress, resulting in part from an increase in COX-2 expression (11).

Mediation of dilation to flow in arterioles of NO-deficient OVE rats.

l-NAME-treated OVX rats that have received 17β-estradiol for 4 wk exhibited an EDHF-mediated dilation to flow, restoring a female pattern of mediation (13, 31). It was previously reported that in cerebral arterioles metabolites of CYP cause vasodilation via activation of COX (7). The present finding that Indo did not but MCZ and ChTX did inhibit flow-induced dilation (Figs. 3 and 4A) argues against the idea that the COX pathway is a downstream effector of the responses, suggesting rather that arteriolar hyperpolarization, consequent to the opening of Ca2+-dependent K+ channels of smooth muscle, is responsible for the full expression of the response.

A number of recent studies have reported gender differences in the regulation of vascular responses to vasoactive agents (2, 5, 13,21, 30, 31). In physiological conditions, estrogen alters the relative contributions of NO and prostaglandins to endothelium-dependent vasodilation through a mechanism that enhances the contribution of NO and mitigates the contribution of prostaglandins. For instance, a histamine receptor agonist-induced, endothelium-dependent dilation of mesenteric arteries was predominantly Indo sensitive in OVX rats, whereas estrogen replacement switched the mediation to be NO dependent (5), suggesting that estrogen modulates the interactions between the NO synthase and COX pathways. Indeed, the gender difference in endothelium-dependent responses of porcine coronary arteries has been found to be dependent on constrictor prostanoids, the synthesis of which is stimulated by male hormones (2). In addition, it was recently reported that estrogen inhibited COX-2 mRNA expression in cultured bovine chondrocytes (24). All of the aforementioned findings could explain why upregulation of COX-2 in response to the loss of NO does not occur in OVE but does occur in OVX rats. On the other hand, the evidence that estrogen favors the contribution of EDHF in the mediation of agonist- and flow-induced responses, especially when NO activity is compromised (8, 13, 22, 25, 30, 31), further supports our conclusion that flow-induced dilation in OVE rats is EDHF dependent.

In summary, we demonstrate that, in NO deficiency, OVX rats exhibit a phenotypic change in their arteriolar flow-dependent responses, which is characteristic of vessels from males. Estrogen replacement therapy reverses the mediation of flow-induced dilation by prostaglandins to one by EDHF, a response that is also present in NO-deficient, intact female rats. These results may be of pathophysiological significance for the estrogen-dependent regulation of vascular function in the absence of or reduction in NO synthesis and may also reveal a unique mechanism by which estrogen evokes EDHF-dependent shear stress-sensitive regulation of skeletal muscle arterioles.

Acknowledgments

This study was supported by American Heart Association Grant 9930244N and National Heart, Lung, and Blood Institute Grants HL-43023 and HL-46813.

Footnotes

Original submission in response to a special call for papers on “Genome and Hormones: Gender Differences in Physiology.”

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